title: Separation optimization of long porous-layer open-tubular columns for nano-LC-MS of limited proteomic samples.
abstract: The single-run resolving power of current 10 μm id porous-layer open-tubular (PLOT) columns has been optimized. The columns studied had a poly(styrene-co-divinylbenzene) porous layer (~0.75 μm thickness). In contrast to many previous studies that have employed complex plumbing or compromising set-ups, SPE-PLOT-LC-MS was assembled without the use of additional hardware/noncommercial parts, additional valves or sample splitting. A comprehensive study of various flow rates, gradient times, and column length combinations was undertaken. Maximum resolution for <400 bar was achieved using a 40 nL/min flow rate, a 400 min gradient and an 8 m long column. We obtained a 2.3-fold increase in peak capacity compared to previous PLOT studies (950 versus previously obtained 400, when using peak width = 2σ definition). Our system also meets or surpasses peak capacities obtained in recent reports using nano-ultra-performance LC conditions or long silica monolith nanocolumns. Nearly 500 proteins (1958 peptides) could be identified in just one single injection of an extract corresponding to 1000 BxPC3 beta catenin (-/-) cells, and ~1200 and 2500 proteins in extracts of 10,000 and 100,000 cells, respectively, allowing detection of central members and regulators of the Wnt signaling pathway.
authors.affiliation: Department of Chemistry, University of Oslo, Blindern, Oslo, Norway. magnus.rogeberg@kjemi.uio.no
authors.forename: Magnus
authors.initials: M
authors.lastname: Rogeberg
authors.forename: Tore
authors.initials: T
authors.lastname: Vehus
authors.forename: Lene
authors.initials: L
authors.lastname: Grutle
authors.forename: Tyge
authors.initials: T
authors.lastname: Greibrokk
authors.forename: Steven Ray
authors.initials: SR
authors.lastname: Wilson
authors.forename: Elsa
authors.initials: E
authors.lastname: Lundanes
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid / Q000295:instrumentation*; D006801:Humans; D040901:Proteomics; D051381:Rats; D053719:Tandem Mass Spectrometry / Q000295:instrumentation*
keywords: Nano-LC; Nanospray; Peak capacity; Porous layer open tubular columns; Wnt pathway